Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.     

Consequences of N-acylation on structure and membrane binding properties of dermaseptin derivative K4-S4-(1-13)

Acyl conjugation to antimicrobial peptides is known to enhance antimicrobial properties. Here, we investigated the consequences of aminolauryl (NC(12)) conjugation to the dermaseptin derivative K(4)-S4-(1-13) (P) on binding properties to bilayer models mimicking bacterial plasma membrane, which is often cited as the ultimate site of action. Isothermal titration calorimetry revealed that acylation was responsible for enhancing the binding affinity of NC(12)-P compared with P (K = 13 x 10(5) and 1.5 x 10(5) m(-1), respectively). Surface plasmon resonance measurements confirmed the isothermal titration calorimetry results (K(app) = 12.6 x 10(5) and 1.53 x 10(5) m(-1), respectively) and further indicated that enhanced adhesion affinity (K(adhesion) = 3 x 10(5) and 1 x 10(5) m(-1), respectively) was coupled to enhanced tendency to insert within the bilayer (K(insertion) = 4.5 and 1.5, respectively). To gain insight into the molecular basis for these observations, we investigated the three-dimensional structures in the presence of dodecylphosphocholine using NMR. The ensemble of NMR-calculated structures (backbone root mean square deviation <0.6 A) showed that the acyl moiety was responsible for a significant molecular reorganization, possibly affecting the electrostatic potential distribution in NC(12)-P relative to that of P. The combined data present compelling evidence in support of the hypothesis that N-acylation affects antimicrobial properties by modifying the secondary structure of the peptide in a manner that facilitates contact with the membrane and consequently increases its disruption.     

Identification of antimicrobial peptide regions derived from genomic sequences of phage lysins

This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysins. Using two lysins (D3 and PhiKZ) we selected and produced two putative peptides (X and Z, respectively) believed to possess antimicrobial properties based on their physicochemical characteristics. The data presented support this hypothesis in that the peptides and various analogs displayed antibacterial activity, bacteriostatic or bactericidal, either individually or upon combination. These putative peptides are believed to act by a mechanism of action resembling that of conventional antimicrobial peptides when judged by both structural and functional criteria. Thus, the peptides are shown to have the ability to form a helical structure, to bind to model bacterial membranes and permeabilize model liposomes. They also display rapid bactericidal kinetics and their antibacterial potency is increased upon amidation. The possible relevance of these results in contributing to potency of phage lysins is discussed. Such peptides may be used to design new potent antimicrobial compounds much needed in face of the ever threatening drug resistance problems.     

Feline lower esophageal sphincter sling and circular muscles have different functional inhibitory neuronal responses

The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO) and a low-tone sling muscle that contracts vigorously to cholinergic stimulation but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and circular muscle responses. Motor responses were induced by electrical field stimulation (EFS; 1-30 Hz) of muscle strips from sling and circular regions of the feline LES in the presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition [NG-nitro-L-arginine (L-NNA)+/-atropine]. This study showed the following. First, sling muscle developed less intrinsic resting tone compared with circular muscle. Second, with EFS, sling muscle contracted (most at 50% by 5 Hz. Third, on neural blockade with atropine or L-NNA+/-atropine, 1) sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition and 2) circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). Fourth, the sling, precontracted with bethanecol, could relax with NO and some non-NO inhibition. Finally, the tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders.     

Phage Therapy of Coral White Plague Disease: Properties of Phage BA3

The bacteriophage BA3 multiplies in and lyses the coral pathogen Thalassomonas loyana. The complete genome of phage BA3 was sequenced; it contains 47 open reading frames with a 40.9% G + C content. Phage BA3 adsorbed to its starved host in seawater with a k = 1.0 × 10⁻⁶ phage ml⁻¹ min⁻¹. Phage therapy of coral disease in aquarium experiments was successful when the phage was added at the same time as the pathogen or 1 day later, but failed to protect the coral when added 2 days after bacterial infection. When the phages were added 1 day after coral infection, the phage titer increased about 100-fold and remained present in the aquarium water throughout the 37-day experiment. At the end of the experiment, the concentration of phages associated with the corals was 2.5 ± 0.5 × 10⁴ per cm² of coral surface. Corals that were infected with the pathogen and treated with phage did not transmit the disease to healthy corals.     

Antimicrobial peptide mimics for improved therapeutic properties

The relatively recent recognition of the major role played by antimicrobial peptides (AMPs) in sustaining an effective host response to immune challenges was greatly influenced by studies of amphibian peptides. AMPs are also widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. However, the peptide formula per se, represents less than ideal drug candidates, namely because of poor bioavailability issues, potential immunogenicity, optional toxicity and high production costs. To address these issues, synthetic peptides have been designed, reproducing the critical peptide biophysical characteristic in unnatural sequence-specific oligomers. Thus, the use of peptidomimetics to overcome the limitations inherent to peptides physical characteristics is becoming an important and promising approach for improving the therapeutic potential of AMPs. Here, we review most recent advances in the design strategies and the biophysical properties of the main classes of mimics to natural AMPs, emphasizing the importance of structure-activity relationship studies in fine-tuning of their physicochemical attributes for improved antimicrobial properties.     

Nuclear transport factors in neuronal function

Active nucleocytoplasmic transport of macromolecules requires soluble transport carriers of the importin/karyopherin superfamily. Although the nuclear transport machinery is essential in all eukaryotic cells, neurons must also mobilise importins and associated proteins to overcome unique spatiotemporal challenges. These include switches in importin α subtype expression during neuronal differentiation, localized axonal synthesis of importin β1 to coordinate a retrograde injury signaling complex on axonal dynein, and trafficking of regulatory and signaling molecules from synaptic terminals to cell bodies. Targeting of RNAs encoding critical components of the importins complex and the Ran system to axons allows sophisticated local regulation of the system for mobilization upon need. Finally, a number of importin family members have been associated with mental or neurodegenerative diseases. The extended roles recently discovered for importins in the nervous system might also be relevant in non-neuronal cells, and the localized modes of importin regulation in neurons offer new avenues to interrogate their cytoplasmic functions.     

Dasyatispora levantinae gen. et sp. nov., a new microsporidian parasite from the common stingray Dasyatis pastinaca in the eastern Mediterranean

A new microsporidian infecting the Mediterranean common stingray Dasyatis pastinaca (Linnaeus, 1758) is described from Iskenderun Bay, Turkey. The parasite invades the disc muscles, producing slender, spindle-shaped subcutaneous swellings that develop into massive, elongated, tumor-like protuberances measuring up to 11 x 4 cm. Severity of the infection may vary from light (1 or 2 small lesions) to intense, with large parts of the dorsal surface covered with lumps and protrusions. These masses contained a yellowish-white caseous substance consisting of degraded host tissue and microsporidian sporophorous vesicles, which in turn contained developing sporonts, sporoblasts and spores. The ripe spore contained a uni-nucleate sporoplasm and large posterior vacuole, and measured 3.8-4.3 x 2.6-2.8 microm. Infection prevalence was 21% in a sample of 143 host individuals examined. All the infected stingray individuals were within the weight class of 300 to 800 g (200 to 305 mm disc width). Phylogenetic analyses of rDNA sequences indicate that this microsporidian belongs to the Pleistophoridae and clusters with species of the genera Ovipleistophora Pekkarinen, Lom & Nilsen, 2002 and Heterosporis Schubert, 1969. However, the morphology, development and host differ distinctly from all reported species, including those belonging to these 2 genera, and it is thus assigned to a newly erected genus and named Dasyatispora levantinae gen. et sp. nov. This is the first record of a microsporidian infection in a batoid. It is also the first microsporidian species to be formally described from an elasmobranch.     

The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction

We show that phage lysogenization, lysogens, and prophage induction are all targeted by CRISPR. The results demonstrate that genomic DNA is not immune to the CRISPR system, that the CRISPR system does not require noncytoplasmic elements, and that the system protects from phages entering and exiting the lysogenic cycle.